Stephanie Speck

High prevalence of genetically diverse Borrelia bavariensis-like strains in Ixodes persulcatus from Selenge Aimag, Mongolia.

In Mongolia, Lyme borreliosis was first reported in 2003. To determine which Borrelia species may contribute to the occurrence of Lyme borreliosis in Mongolia, real-time PCR was conducted on 372 adult Ixodes persulcatus ticks collected in Selenge Aimag, the province with the highest incidence of human Lyme borreliosis. 24.5% of ticks were identified to be positive for Borrelia burgdorferi sensu lato DNA. Species differentiation using an SNP-based real-time PCR and multi-locus sequence analysis revealed that strains phylogenetically closely related to B. bavariensis (previously known as B. garinii OspA serotype 4) is the most prevalent species, showing an unexpectedly high genetic diversity.

Rickettsia raoultii, the predominant Rickettsia found in Mongolian Dermacentor nuttalli

Since the year 2005, clinical patterns resembling tick-borne rickettsioses have been noticed in Mongolia. Epidemiological data regarding species of the aetiological agent, tick vector, prevalence, and distribution as well as incidence of human cases throughout Mongolia are still sparse to date. In order to identify Rickettsia species occurring in Mongolia, we investigated Dermacentor nuttalli (n=179) and Ixodes persulcatus (n=374) collected in 4 selected provinces. Rickettsia raoultii was the predominant Rickettsia (82% prevalence) found in D. nuttalli and was also detected in I. persulcatus (0.8%). The Rickettsia prevalence in D. nuttalli from different provinces varied between 70% and 97%. In addition, R. sibirica was identified in approximately 4% of D. nuttalli, but solely from Arkhanghai province. The results of this study extend the common knowledge about the geographic distribution of R. raoultii and its high prevalence in D. nuttalli. Although the pathogenicity of this Rickettsia is still unclear, it should be considered in Mongolian patients suspected of having tick-borne rickettsiosis.

Detection of Rickettsia helvetica in ticks collected from European hedgehogs (Erinaceus europaeus, Linnaeus, 1758).

The role of wild mammals in the dissemination and maintenance of Rickettsia in nature is still under investigation. European hedgehogs (Erinaceus europaeus) are often heavily infested by tick and flea species that are known to harbor and transmit different Rickettsia spp. We investigated ixodid ticks sampled from European hedgehogs for the presence of Rickettsia. A total of 471 Ixodes ricinus and 755 I. hexagonus were collected from 26 German and 7 British European hedgehogs. These were tested by a genus-specific real-time PCR assay targeting the citrate synthase gene (gltA). The rickettsia minimum infection rate was 11.7% with an increase detected with each parasitic tick stage. No significant difference in Rickettsia prevalence in the 2 Ixodes species was detected. Using sequencing of partial ompB, Rickettsia helvetica was the only species identified. More than half of the hedgehogs carried Rickettsia-positive ticks. In addition, tissue samples from 2/5 hedgehogs (where tissue DNA was available) were PCR-positive. These results show that European hedgehogs are exposed to R. helvetica via infected ticks and might be involved in the natural transmission cycle of this Rickettsia species.

Effects of Cationic Antimicrobial Peptides on Liquid-Preserved Boar Spermatozoa

Antibiotics are mandatory additives in semen extenders to control bacterial contamination. The worldwide increase in resistance to conventional antibiotics requires the search for alternatives not only for animal artificial insemination industries, but also for veterinary and human medicine. Cationic antimicrobial peptides are of interest as a novel class of antimicrobial additives for boar semen preservation. The present study investigated effects of two synthetic cyclic hexapeptides (c-WFW, c-WWW) and a synthetic helical magainin II amide derivative (MK5E) on boar sperm during semen storage at 16°C for 4 days. The standard extender, Beltsville Thawing Solution (BTS) containing 250 µg/mL gentamicin (standard), was compared to combinations of BTS with each of the peptides in a split-sample procedure. Examination revealed peptide- and concentration-dependent effects on sperm integrity and motility. Negative effects were more pronounced for MK5E than in hexapeptide-supplemented samples. The cyclic hexapeptides were partly able to stimulate a linear progressive sperm movement. When using low concentrations of cyclic hexapeptides (4 µM c-WFW, 2 µM c-WWW) sperm quality was comparable to the standard extender over the course of preservation. C-WFW-supplemented boar semen resulted in normal fertility rates after AI. In order to investigate the interaction of peptides with the membrane, electron spin resonance spectroscopic measurements were performed using spin-labeled lipids. C-WWW and c-WFW reversibly immobilized an analog of phosphatidylcholine (PC), whereas MK5E caused an irreversible increase of PC mobility. These results suggest testing the antimicrobial efficiency of non-toxic concentrations of selected cyclic hexapeptides as potential candidates to supplement/replace common antibiotics in semen preservation.

Influence of clinical and laboratory variables on faecal antigen ELISA results in dogs with canine parvovirus infection.

False negative faecal canine parvovirus (CPV) antigen ELISA results in dogs with CPV infection are common, but the factors that lead to these false negative results are still unknown. The aim of this study was to investigate whether dogs with a false negative faecal CPV antigen ELISA result have milder clinical signs and laboratory changes, a lower faecal virus load, higher faecal and serum CPV antibody titres and a faster recovery than dogs with a positive result. Eighty dogs with CPV infection, confirmed by the presence of clinical signs and a positive faecal CPV polymerase chain reaction (PCR), were assigned to two groups according to their faecal antigen ELISA result. Time until presentation, severity of symptoms, laboratory parameters, faecal virus load, faecal and serum antibody titres, and CPV sequencing data were compared between both groups. In 38/80 dogs that were hospitalised until recovery, the time to recovery, mortality, and the course of the disease were compared between dogs with positive and negative faecal antigen ELISA results. Of the 80 dogs included, 41 (51.3%) had a false negative faecal antigen ELISA result. ELISA-negative dogs had a significantly shorter time until presentation, lower frequency of defaecation, lower faecal virus load, and higher serum antibody concentrations than ELISA-positive dogs. Laboratory changes, CPV shedding, and outcomes were not associated with faecal antigen ELISA results. In conclusion, low faecal CPV load and antibodies binding to CPV antigen in faeces are likely to be important reasons for false negative faecal antigen ELISA results. Dogs with clinical signs of CPV infection should be retested by faecal PCR.

Proposal of Vespertiliibacter pulmonis gen. nov., sp. nov. and two genomospecies as new members of the family Pasteurellaceae isolated from European bats.

Five bacterial strains isolated from bats of the family Vespertilionidae were characterized by phenotypic tests and multilocus sequence analysis (MLSA) using the 16S rRNA gene and four housekeeping genes (rpoA, rpoB, infB, recN). Phylogenetic analyses of individual and combined datasets indicated that the five strains represent a monophyletic cluster within the family Pasteurellaceae. Comparison of 16S rRNA gene sequences demonstrated a high degree of similarity (98.3-99.9%) among the group of bat-derived strains, while searches in nucleotide databases indicated less than 96% sequence similarity to known members of the Pasteurellaceae. The housekeeping genes rpoA, rpoB, infB and recN provided higher resolution compared with the 16S rRNA gene and subdivided the group according to the bat species from which the strains were isolated. Three strains derived from noctule bats shared 98.6-100% sequence similarity in all four genes investigated, whereas, based on rpoB, infB and recN gene sequences, 91.8-96% similarity was observed with and between the remaining two strains isolated from a serotine bat and a pipistrelle bat, respectively. Genome relatedness as deduced from recN gene sequences correlated well with the results of MLSA and indicated that the five strains represent a new genus. Based on these results, it is proposed to classify the five strains derived from bats within Vespertiliibacter pulmonis gen. nov., sp. nov. (the type species), Vespertiliibacter genomospecies 1 and Vespertiliibacter genomospecies 2. The genus can be distinguished phenotypically from recognized genera of the Pasteurellaceae by at least three characteristics. All strains are nutritionally fastidious and require a chemically defined supplement with NAD for growth. The DNA G+C content of strain E127/08(T) is 38.2 mol%. The type strain of Vespertiliibacter pulmonis gen. nov., sp. nov. is E127/08(T) ( = CCUG 64585(T) = DSM 27238(T)). The reference strains of Vespertiliibacter genomospecies 1 and 2 are E145/08 and E157/08, respectively.

An inactivated whole-virus porcine parvovirus vaccine protects pigs against disease but does not prevent virus shedding even after homologous virus challenge

Inactivated whole-virus vaccines against porcine parvovirus (PPV) can prevent disease but not infection and virus shedding after heterologous virus challenge. Here, we showed that the same is true for a homologous challenge. Pregnant sows were vaccinated with an experimental inactivated vaccine based on PPV strain 27a. They were challenged on day 40 of gestation with the virulent porcine parvovirus PPV-27a from which the vaccine was prepared (homologous challenge). On day 90 of gestation, the fetuses from vaccinated sows were protected against disease, while the fetuses of the non-vaccinated sows (control group) exhibited signs of parvovirus disease. All gilts, whether vaccinated or not vaccinated, showed a boost of PPV-specific antibodies indicative of virus infection and replication. Low DNA copy numbers, but not infectious virus, could be demonstrated in nasal or rectal swabs of immunized sows, but high copy numbers of challenge virus DNA as well as infectious virus could both be demonstrated in non-vaccinated sows.

Faecal shedding of canine parvovirus after modified-live vaccination in healthy adult dogs

Since little is known about the persistence and faecal shedding of canine parvovirus (CPV) in dogs after modified-live vaccination, diagnostic tests for CPV can be difficult to interpret in the post-vaccination period. The primary aim of this study was to determine the incidence, duration and extent of CPV vaccine virus shedding in adult dogs and to investigate related factors, including the presence of protective antibodies, increase in anti-CPV antibody titres and development of any gastrointestinal side-effects. A secondary objective was to assess prevalence of CPV field virus shedding in clinically healthy dogs due to subclinical infections. One hundred adult, healthy privately owned dogs were vaccinated with a commercial CPV-2 modified-live vaccine (MLV). Faeces were tested for the presence of CPV DNA on days 0 (prior to vaccination), 3, 7, 14, 21 and 28 by quantitative real-time PCR. Pre- and post-vaccination serum titres were determined by haemagglutination inhibition on days 0, 7 and 28. Transient excretion of CPV DNA was detected in 2.0% of dogs before vaccination. About one quarter of dogs (23.0%) shed CPV DNA during the post-vaccination period, but field and vaccine virus differentiation by VP2 gene sequencing was only successful in few samples. Faecal CPV excretion occurred despite protective serum antibody titres. Post-vaccination CPV shedding was not related to adequate antibody response after vaccination or to the occurrence of gastrointestinal side-effects. Despite individual differences, CPV DNA was detectable for up to 28 days after vaccination, although the faecal CPV DNA load in these clinically healthy dogs was very low.

Detection of Babesia venatorum, Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis in Ixodes persulcatus ticks from Mongolia.

Information about the prevalence and geographical distribution of tick-borne pathogens Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis, and Babesia spp. is still rare in Mongolia. We tested 275 Ixodes persulcatus ticks for A. phagocytophilum, Cand. N. mikurensis and Babesia spp. and 125 Dermacentor nuttalli ticks especially for Babesia spp. using different PCR methods. Ticks were collected from three provinces (Selenge, Arkhangai, Khentii) in Mongolia. DNA of A. phagocytophilum, Cand. N. mikurensis and Babesia spp. were found with a prevalence of 6.2%, 1.5% and 3.3% in each case in I. persulcatus ticks. This is the first time Cand. N. mikurensis was found in ticks from Mongolia. Sequence analysis of Babesia spp.-positive amplicons showed exclusively B. venatorum, which had also not been mentioned in Mongolia before. On the contrary, all D. nuttalli ticks tested negatively for Babesia spp. This study demonstrates that all three zoonotic pathogens are present in I. persulcatus ticks in Mongolia, and justify the need for further investigations of a more detailed genetic characterization of these pathogens.

Survey for hantaviruses, tick-borne encephalitis virus, and Rickettsia spp. in small rodents in Croatia.

In Croatia, several rodent- and vector-borne agents are endemic and of medical importance. In this study, we investigated hantaviruses and, for the first time, tick-borne encephalitis virus (TBEV) and Rickettsia spp. in small wild rodents from two different sites (mountainous and lowland region) in Croatia. In total, 194 transudate and tissue samples from 170 rodents (A. flavicollis, n=115; A. agrarius, n=2; Myodes glareolus, n=53) were tested for antibodies by indirect immunofluorescence assays (IIFT) and for nucleic acids by conventional (hantaviruses) and real-time RT-/PCRs (TBEV and Rickettsia spp.). A total of 25.5% (24/94) of the rodents from the mountainous area revealed specific antibodies against hantaviruses. In all, 21.3% (20/94) of the samples from the mountainous area and 29.0% (9/31) from the lowland area yielded positive results for either Puumala virus (PUUV) or Dobrava-Belgrade virus (DOBV) using a conventional RT-PCR. All processed samples (n=194) were negative for TBEV by IIFT or real-time RT-PCR. Serological evidence of rickettsial infection was detected in 4.3% (4/94) rodents from the mountainous region. Another 3.2% (3/94) rodents were positive for Rickettsia spp. by real-time PCR. None of the rodents (n=76) from the lowland area were positive for Rickettsia spp. by real-time PCR. Dual infection of PUUV and Rickettsia spp. was found in one M. glareolus from the mountainous area by RT-PCR and real-time PCR, respectively. To our knowledge, this is the first detection of Rickettsia spp. in small rodents from Croatia. Phylogenetic analyses of S- and M-segment sequences obtained from the two study sites revealed well-supported subgroups in Croatian PUUV and DOBV. Although somewhat limited, our data showed occurrence and prevalence of PUUV, DOBV, and rickettsiae in Croatia. Further studies are warranted to confirm these data and to determine the Rickettsia species present in rodents in these areas.